Device for focusing a microscope objective on a sample

ABSTRACT

A device for focusing a microscope objective on a sample accurately with a high spatial resolution. The device has a positioning unit having a main body, an objective holder movably supported on the main body and adapted to hold the microscope objective, and an actuator for moving the objective holder along the optical axis of the microscope objective. The objective holder holds the microscope objective only at a front portion of the microscope objective facing the sample. The positioning unit includes one or more lever arms, each of which coupled at its one end via a first flexure bearing to the main body and at its other end via a second flexure bearing to the objective holder. The main body of the positioning unit is attached to a stage that carries the sample.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority of the German patent application DE102010060841.6 having a filing date of Nov. 26, 2010. The entire contentof this prior German patent application 102010060841.6 is herewithincorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates to a device for focusing a microscopeobjective on a sample, the device including a positioning unit having amain body, an objective holder movably supported on the main body andadapted to hold the microscope objective, and an actuator for moving theobjective holder along the optical axis of the microscope objective.

In high-resolution light microscopy, it is particularly important thatthe microscope objective be held in a stable and precise position in theimaging beam path. For example, the spatial resolutions to be achievedin modern light microscopic methods are desired to be below 50 nm in alateral direction; i.e., perpendicular to the optical axis of themicroscope objective. Therefore, unintentional relative lateralmovements between the sample and the microscope objective, which mayresult, for example, from temperature fluctuations, are to be avoided tothe extent possible. This holds especially true for a number of methodswhich use imaging times of several minutes, during which even thesmallest displacements of the microscope objective perpendicular to theoptical axis would together result in image offset and, thus, in acorrespondingly reduced lateral resolution.

In addition, in the light microscopic methods of the aforementionedtype, the microscope objective must be focused on the sample withparticularly high accuracy. The travel of the microscope objective alongthe optical axis is typically on the order of about 100 μm. Among thepositioning units used in microscopes to cause the focusing movement ofthe microscope objective along the optical axis, increasing use is madeof high-precision MEMS devices which include a main body, an objectiveholder movably supported on the main body, and an (for example,piezoceramic) actuator for precisely moving the objective holder, andthus microscope objective, along the optical axis so as to focus themicroscope objective on the sample.

A conventional microscope objective is usually threaded at its rear end;i.e., the end facing away from the sample. This thread is used, forexample, to screw the microscope objective into a commercially availableobjective turret. When using a positioning unit of the aforementionedtype, this microscope thread is used to screw the microscope objectiveat its rear end into the objective holder, possibly with a suitableadapter positioned therebetween.

In the prior art, there are known various other approaches which aim toensure that the microscope objective is held in as stable a position aspossible and focused with high accuracy. For example, U.S. PatentApplication No. 2002/0015225 describes a microscope in which the sampleholder and the focusing unit are mounted directly on the microscopeobjective. However, this has the disadvantage that easy changing ofmicroscope objectives is no longer possible. Moreover, manipulation ofthe sample has a direct effect on the focusing unit, which may impairthe focusing accuracy.

European Patent Application EP 1 418 456 A1 describes a microscope inwhich unwanted deviations of the position of the microscope objectiveperpendicular to the optical axis are to be prevented by a rotationallysymmetrical configuration around the optical axis and the use of opticalcomponents whose thermal expansion coefficients are suitably matched toeach other.

International Patent Application published under the publication numberWO 2006/056178 A1 discloses a microscope objective which is integratedinto a sample stage by means of a guide sleeve. However, while this doesincrease the stability along the optical axis, there is a not negligibleplay of the microscope objective within the guide sleeve in a directionperpendicular to the optical axis.

U.S. Pat. No. 6,731,327 B1 describes a positioning unit for moving amicroscope objective along the optical axis. This positioning unit has aclamping device that additionally stabilizes the microscope objective inits operating position. However, providing the clamping device makesthis a relatively complex design.

From the German Patent Application published under the publicationnumber DE 199 49 044 A1 there is known a device for fine positioning ofa microscope objective using a double parallel spring element. Thedouble parallel spring element acts on the end of the microscopeobjective that faces away from the sample, as a result of which theopposite free end of the microscope objective facing the sample may tiltto a not negligible degree, leading to unwanted image offset and acorrespondingly reduced spatial resolution.

From the U.S. patent application published under the publication numberUS 2009/0284853 A1 a focusing device is known, comprising a pair of leafsprings, each of which is coupled with one end to a main body and withthe other end to an objective holder. The objective holder is formed byan external thread arranged on the outer surface of the lens barrel anda nut engaged with the external thread.

In the U.S. patent application published under the publication number US2010/0091363 A1 an objective holder is described, with which one of aplurality of objectives can be selectively brought into the microscopebeam path. The plurality of objectives respectively have a contactsurface arranged along the optical axis at the same height, whichcontacts a corresponding, fixed contact surface, so that parfocalitybetween the various objectives is provided.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a device that allowsa microscope objective to be accurately focused on a sample with a highspatial resolution.

According to an aspect of the invention, this object is achieved by adevice for focusing a microscope objective on a sample, comprising: apositioning unit having a main body, an objective holder movablysupported on the main body and adapted to hold the microscope objective,and an actuator for moving the objective holder along the optical axisof the microscope objective; wherein the objective holder holds themicroscope objective only at a front portion of the microscope objectivefacing the sample; the positioning unit includes one or more lever arms,each of which coupled at its one end via a first flexure bearing to themain body and at its other end via a second flexure bearing to theobjective holder; and the main body of the positioning unit is attachedto a stage that carries the sample.

DETAILED DESCRIPTION OF THE INVENTION

Thus, in contrast to the conventional systems where the microscopeobjective is held at the rear end; i.e., the end facing away from thesample (and usually by the thread provided at that end), it is a featureof the present invention that the microscope objective is held at afront portion facing the sample, which is where the so-called front lensis located. This is because it has been found that stabilizing theobjective at its front end, as proposed by the present invention, hasadvantages over the conventional method of stabilizing it at its rearend when it comes to achieving the highest possible level of lateralresolution.

In a microscope objective, there is always a tilt-tolerant planeperpendicular to the optical axis, in which tilting of the microscopeobjective perpendicular to the optical axis does, at least in a firstapproximation, not result in image offset. This plane coincides with thefirst principal plane of the objective optical system. The exactposition of the plane depends on the specific design of the microscopeobjective, and especially on the specific arrangement and number oflenses in the objective. However, as for a high-magnification microscopeobjective used in high-resolution microscopy, which, having a totallength of about 1.7 Inch to 2.6 Inch (about 45 to 65 mm), has a focallength of only a few millimeters, it can definitely be said that thetilt-tolerant plane lies in the front portion of the objective, whichfaces the sample. This front portion contains the above-mentioned frontlens as the first lens facing the sample, as viewed from the sample.Therefore, in a practical implementation of the present invention, inorder to significantly improve the lateral resolution, it will in allcases be sufficient that the objective holder engage the microscopeobjective as far forward as possible under the given conditions.

The positioning unit has one or more lever arms, each of which iscoupled at its one end via a first flexure bearing to the main body andat its other end via a second flexure bearing to the objective holder.In this embodiment, the positioning unit is a single-piece component,the rigid portions of which are constituted by the main body and theobjective holder and are movable relative to each other by way offlexure bearings. The flexure bearings are each constituted by a portionof the component that has a reduced bending resistance compared toadjacent portions. This reduced bending resistance may be achieved, forexample, by locally reducing the cross section of the materialconstituting the component.

In an advantageous embodiment, two lever arms are arranged parallel toone another and, together with the main body and the objective holder,form a parallelogram configuration. Such a parallelogram configurationenables a precise positioning movement of the objective holder along theoptical axis with very little unwanted movement perpendicular to theoptical axis. To this end there is provided the actuator, which exerts aforce on the lever arms, causing a controlled movement of theparallelogram configuration.

The main body of the positioning unit is attached to a stage whichcarries the sample. In this embodiment, the front portion of themicroscope objective, which faces the sample, is coupled via thepositioning unit to the sample placed on the stage. This advantageouslyresults in a short coupling distance between the microscope objectiveand the sample, which avoids excessive relative movement between themicroscope objective and the sample, particularly relative movementcaused by temperature changes.

In another advantageous embodiment, the actuator is a piezoceramicelement which is integrated into the main body of the positioning unit.Such a piezoceramic element is capable of very precisely moving theobjective holder along the optical axis.

Preferably, the objective holder includes a ring having an internalthread, and the microscope objective has an external thread on at itsfront portion facing the sample, the internal thread of the ring beingthreaded onto said external thread. This holding arrangement for themicroscope objective, which is rotationally symmetric with respect tothe optical axis, further reduces displacements perpendicular to theoptical axis.

In accordance with a further aspect of the present invention, there isprovided a microscope having a device for focusing a microscopeobjective.

The present invention is described below in more detail with referenceto an exemplary embodiment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a high-resolution light microscope constituting anexemplary embodiment of the present invention.

DETAILED DESCRIPTION OF THE DRAWINGS

The light microscope generally designated 10 in the FIG. 1 includes aninverted stand 12, on which is placed a stage 14 having an opening 16extending therethrough. A sample 18 is placed on stage 14 in such a waythat it covers opening 16. A positioning unit 20 is attached to theunderside of stage 14.

Positioning unit 20 includes a main body 22 and an objective holder 24movably supported on main body 22. A microscope objective 26 is held onobjective holder 24. A piezoceramic actuator 27 integrated into mainbody 22 and shown only schematically in the FIG. 1 is capable of movingobjective holder 24 along an optical axis O so as to, in turn, moveobjective 26 along optical axis O in order to focus it on sample 18.Actuator 27 is connected to a control unit 29. The control signals ofsaid control unit are used to generate the movements of actuator 27,which in turn move objective holder 24. In the present exemplaryembodiment, control unit 29 is located outside of stand 12. However, itmay also be integrated into stand 12.

The following first describes positioning unit 20 in detail.

Objective holder 24 is movably supported on main body 22 by lever arms28 and 30. In the present exemplary embodiment, positioning unit 20 is asingle-piece component, whose elements, namely main body 22, objectiveholder 24 as well as lever arms 28 and 30, are movable relative to eachother and separated from one another by cavities 32 formed in thematerial. Cavities 32 are configured in positioning unit 20 in such away that in the region of the longitudinal ends of lever arms 28 and 30,the material constituting positioning unit 20 has regions of locallyreduced cross section defining flexure bearings. For the sake ofclarity, only one of the flexure bearings is denoted by referencenumeral 34 in the FIG. 1.

Due to the aforementioned regions of locally reduced cross section, thebending resistance of flexure bearings 34 in a direction parallel tooptical axis O is significantly reduced compared to the bendingresistance transverse to optical axis O. Because of this reduced bendingresistance, flexure bearings 34 enable objective holder 24 to be movedrelative to main body 22 along optical axis O by way of the lever arms28 and 30. Thus, objective holder 24, main body 22 and the two leverarms 28 and 30 together form a parallelogram-type configuration. Thefixed base of the parallelogram is provided by main body 22, while thethree remaining movable sides of the parallelogram are formed byobjective holder 24 and the two lever arms 28 and 30.

In this connection, it should be noted that the magnitude of thepositioning movement of objective holder 24 along optical axis O is nogreater than about 100 μm. At this very small positioning movement, theunintentional movement of objective holder 24 perpendicular to opticalaxis O, which is caused by the above-described parallelogramconfiguration, is of negligible magnitude.

Objective holder 24 is formed by an elongated portion 40 extendingparallel to optical axis O and a ring 42 extending from elongatedportion 40 in a direction perpendicular to optical axis O. Ring 42 hasan internal thread 44, which is rotationally symmetric with respect tooptical axis O. This internal thread 44 is threadedly engaged with anexternal thread 46 formed on an annular collar 48 of microscopeobjective 26. Annular collar 48 is disposed at a front portion ofmicroscope objective 26 that faces sample 18. Accordingly, microscopeobjective 26 is coupled at its front portion to positioning unit 20.

Thus, in the present exemplary embodiment, microscope objective 26 isheld at a position immediately proximate the objective lens thatimmediately faces sample 18. In the Figure, this lens, which is alsoreferred to as “front lens”, is denoted by reference numeral 50. Ofcourse, microscope objective 26 includes additional lenses, which areschematically indicated in the Figure by reference numerals 52 and 54.

Like a conventional objective, microscope objective 26 also has anobjective thread 56 at its rear end; i.e., the end facing away fromsample 18. Objective thread 56 is used, for example, for adjustmentduring manufacture.

Light microscope 10 further has an illumination device 58 including alight source 60 and lenses 62 and 64. The fluorescence excitation lightfrom light source 60 passes through lenses 62 and 64 and is reflected bya dichroic mirror 66 along optical axis O and into objective 26. Thefluorescent light coming from sample 18 passes through front lens 50 andfurther lenses 52 and 54 of microscope objective 26 to semitransparentmirror 66, which allows the fluorescent light to pass therethroughtoward an additional lens 68. The fluorescent light passing through lens68 is finally reflected by a mirror 70 into a camera 72 attached tostand 12.

It will be readily understood that the embodiment described above is forillustrative purposes only. For example, it is also possible to use apositioning unit other than the single-piece unit 20 provided withflexure bearings 34. It is possible to use, for example, a springarrangement which is driven by an external actuator so as to movemicroscope objective 26 along optical axis O and thereby focus it.

Also, objective holder 24 may be coupled to microscope objective 26 in away other than by two meshing threads 44 and 46 as shown in the Figure.What is essential to the present invention is that microscope objective26 is held at its front portion facing sample 18.

LIST OF REFERENCE NUMERALS

-   10 light microscope-   12 stand-   14 stage-   16 opening-   18 sample-   20 positioning unit-   22 main body-   24 objective holder-   26 microscope objective-   27 actuator-   28 lever arm-   29 control unit-   30 lever arm-   32 cavity-   34 flexure bearing-   40 elongated portion-   42 ring-   44 internal thread-   46 external thread-   48 annular collar-   50 front lens-   52 lens-   54 lens-   56 objective thread-   58 illumination device-   60 light source-   62 lens-   64 lens-   66 dichroic mirror-   68 lens-   70 mirror-   72 camera

1. A device for focusing a microscope objective on a sample, comprising:a positioning unit having a main body, an objective holder movablysupported on the main body and adapted to hold the microscope objective,and an actuator for moving the objective holder along the optical axisof the microscope objective; wherein the objective holder holds themicroscope objective only at a front portion of the microscope objectivefacing the sample; the positioning unit includes one or more lever arms,each of which coupled at its one end via a first flexure bearing to themain body and at its other end via a second flexure bearing to theobjective holder; and the main body of the positioning unit is attachedto a stage that carries the sample.
 2. The device as recited in claim 1,further comprising two lever arms that are arranged in parallel to oneanother and form together with the main body and the objective holder aparallelogram configuration.
 3. The device as recited in claim 1,wherein the actuator is a piezoceramic element that is integrated intothe main body of the positioning unit.
 4. The device as recited in claim1, wherein the objective holder includes a ring having an internalthread; and the microscope objective has at its front portion facing thesample an external thread on that the internal thread of the ring isscrewed.
 5. The device as recited in claim 1, wherein the total lengthof the microscope objective along the optical axis is in a range from1.7 Inch to 2.6 Inch; the front portion at which the microscopeobjective is held and that faces the sample has a length in a range from0.1 to 0.4 Inch along the optical axis; and the focal length of themicroscope objective is in a range from 0.1 to 0.2 Inch.
 6. A microscopehaving a device according to claim 1.